Amplifiable adeno-associated virus (AAV) packaging cassettes for the production of recombinant AAV vectors

ABSTRACT

High-efficiency AAV packaging constructs and methods for their use are provided in the present invention. These high-efficiency packaging constructs comprise an activating element (such as the P1 sequence located within the AAV S1 integration site of human chromosome 19) amplifiably linked to one or more AAV packaging genes. The constructs may be either integrated into a mammalian cell genome or maintained episomally. Use of the high-efficiency AAV packaging vectors of the invention provides for controlled amplifiable production of rAAV vector constructs.

TECHNICAL FIELD

This invention is in the field of viral constructs for gene delivery.More specifically, the invention is in the field of recombinant DNAconstructs for use in the production of adeno-associated virus (AAV)vectors for gene delivery.

BACKGROUND

Vectors based on adeno-associated virus (AAV) are believed to haveutility for gene therapy but a significant obstacle has been thedifficulty in generating such vectors in amounts that would beclinically useful for human gene therapy applications. This is aparticular problem for in vivo applications such as direct delivery tothe lung. Another important goal in the gene therapy context, discussedin more detail herein, is the production of vector preparations that areessentially free of replication-competent virions. The followingdescription briefly summarizes studies involving adeno-associated virusand AAV vectors, and then describes a number of novel improvementsaccording to the present invention that are useful for efficientlygenerating high titer recombinant AAV vector (rAAV) preparationssuitable for use in gene therapy.

Adeno-associated virus is a defective parvovirus that grows only incells in which certain functions are provided by a co-infecting helpervirus. General reviews of AAV may be found in, for example, Carter,1989, Handbook of Parvoviruses, Vol. I, pp. 169-228, and Berns, 1990,Virology, pp. 1743-1764, Raven Press, (New York). Examples ofco-infecting viruses that provide helper functions for AAV growth andreplication are adenoviruses, herpesviruses and, in some cases,poxviruses such as vaccinia The nature of the helper function is notentirely known but it appears that the helper virus indirectly rendersthe cell permissive for AAV replication. This belief is supported by theobservation that AAV replication may occur at low efficiency in theabsence of helper virus co-infection if the cells are treated withagents that are either genotoxic or that disrupt the cell cycle.

Although AAV may replicate to a limited extent in the absence of helpervirus, under such conditions as noted above, more generally infection ofcells with AAV in the absence of helper functions results in theproviral AAV genome integrating into the host cell genome. Unlike otherviruses, such as many retroviruses, it appears that AAV generallyintegrates into a unique position in the human genome. Thus, it has beenreported that, in human cells, AAV integrates into a unique position(referred to as an “AAV integration site”) which is located onchromosome 19. See, e.g., Weitzman et al. (1994) Proc. Nat'l. Acad. Sci.USA 91: 5808-5812. If host cells having an integrated AAV aresubsequently superinfected with a helper virus such as adenovirus, theintegrated AAV genome can be rescued and replicated to yield a burst ofinfectious progeny AAV particles. A sequence at the AAV integrationsite, referred to as “P1,” shares homology with the AAV invertedterminal repeat (ITR) sequence, exhibits activity in a cell-freereplication system, and is believed to be involved in both theintegration and rescue of AAV. See, e.g., Weitzman et al., id., Kotin etal. (1992) EMBO J. 11:5071-5078, and Urcelay et al., J. Virol. 69:2038-2046. The fact that integration of AAV appears to be efficient andsite-specific makes AAV a useful vector for introducing genes into cellsfor uses such as human gene therapy.

AAV has a very broad host range without any obvious species or tissuespecificity and can replicate in virtually any cell line of human,simian or rodent origin provided that an appropriate helper is present.AAV is also relatively ubiquitous and has been isolated from a widevariety of animal species including most mammalian and several avianspecies.

AAV is not associated with the cause of any disease. Nor is AAV atransforming or oncogenic virus, and integration of AAV into the geneticmaterial of human cells generally does not cause significant alterationof the growth properties or morphological characteristics of the hostcells. These properties of AAV also recommend it as a potentially usefulhuman gene therapy vector because most of the other viral systemsproposed for this application, such as retroviruses, adenoviruses,herpesviruses, or poxviruses, are disease-causing.

Although various serotypes of AAV are known to exist, they are allclosely related functionally, structurally, and at the genetic level(see, e.g., Blacklow, 1988, pp. 165-174 of Parvoviruses and HumanDisease, J. R. Pattison (ed.); and Rose, 1974, Comprehensive Virology 3:1-61). For example, all AAV serotypes apparently exhibit very similarreplication properties mediated by homologous rep genes; and all bearthree related capsid proteins such as those expressed in AAV2. Thedegree of relatedness is further suggested by heteroduplex analysiswhich reveals extensive cross-hybridization between serotypes along thelength of the genome; and the presence of analogous self-annealingsegments at the termini that correspond to inverted terminal repeats(ITRs). The similar infectivity patterns also suggest that thereplication functions in each serotype are under similar regulatorycontrol. Thus, although the AAV2 serotype was used in variousillustrations of the present invention that are set forth in theExamples, general reference to AAV herein encompasses all AAV serotypes,and it is fully expected that the methods and compositions disclosedherein will be applicable to all AAV serotypes.

AAV particles are comprised of a proteinaceous capsid having threecapsid proteins, VP1, VP2 and VP3, which enclose a DNA genome. The AAV2DNA genome, for example, is a linear single-stranded DNA molecule havinga molecular weight of about 1.5×10⁶ daltons and a length of about 5 kb.Individual particles package only one DNA molecule strand, but this maybe either the “plus” or “minus” strand. Particles containing eitherstrand are infectious and replication occurs by conversion of theparental infecting single strand to a duplex form and subsequentamplification of a large pool of duplex molecules from which progenysingle strands are displaced and packaged into capsids. Duplex orsingle-strand copies of AAV genomes can be inserted into bacterialplasmids or phagemids and transfected into adenovirus-infected cells;these techniques have facilitated the study of AAV genetics and thedevelopment of AAV vectors.

The AAV genome, which encodes proteins mediating replication andencapsidation of the viral DNA, is generally flanked by two copies ofinverted terminal repeats (ITRs). In the case of AAV2, for example, theITRs are each 145 nucleotides in length, flanking a unique sequenceregion of about 4470 nucleotides that contains two main open readingframes for the rep and cap genes (Srivastiva et al., 1983, J. Virol.,45:555-564; Hermonat et al., J. Virol. 51:329-339; Tratschin et al.,1984a, J. Virol., 51:611-619). The AAV2 unique region contains threetranscription promoters p5, p19, and p40 (Laughlin et al., 1979, Proc.Natl. Acad. Sci. USA, 76:5567-5571) that are used to express the rep andcap genes. The ITR sequences are required in cis and are sufficient toprovide a functional origin of replication (ori), signals required forintegration into the cell genome, and efficient excision and rescue fromhost cell chromosomes or recombinant plasmids. It has also been shownthat the ITR can function directly as a transcription promoter in an AAVvector. See Flotte et al., 1993, supra; and Carter et al., U.S. Pat. No.5,587,308.

The rep and cap gene products are required in trans to provide functionsfor replication and encapsidation of viral genome, respectively. Again,using AAV2 for purposes of illustration, the rep gene is expressed fromtwo promoters, p5 and p19, and produces four proteins. Transcriptionfrom p5 yields an unspliced 4.2 kb mRNA encoding a first Rep protein(Rep78), and a spliced 3.9 kb mRNA encoding a second Rep protein(Rep68). Transcription from p19 yields an unspliced mRNA encoding athird Rep protein (Rep52), and a spliced 3.3 kb mRNA encoding a fourthRep protein (Rep40). Thus, the four Rep proteins all comprise a commoninternal region sequence but differ in their amino and carboxyl terminalregions. Only the large Rep proteins (i.e. Rep78 and Rep68) are requiredfor AAV duplex DNA replication, but the small Rep proteins (i.e. Rep52and Rep40) appear to be needed for progeny, single-strand DNAaccumulation (Chejanovsky & Carter, 1989, Virology 173:120-128). Rep68and Rep78 bind specifically to the hairpin conformation of the AAV ITRand possess several enzyme activities required for resolving replicationat the AAV termini. Rep52 and Rep40 have none of these properties.Reports by C. Hölscher et al. (1994, J. Virol. 68:7169-7177; and 1995,J. Virol. 69:6880-6885) have suggested that expression of Rep78 or Rep68 may in some circumstances be sufficient for infectious particleformation.

The Rep proteins, primarily Rep78 and Rep68, also exhibit pleiotropicregulatory activities including positive and negative regulation of AAVgenes and expression from some heterologous promoters, as well asinhibitory effects on cell growth (Tratschin et al., 1986, Mol. Cell.Biol. 6:2884-2894; Labow et al., 1987, Mol. Cell. Biol., 7:1320-1325;Khleif et al., 1991, Virology, 181:738-741). The AAV p5 promoter isnegatively auto-regulated by Rep78 or Rep68 (Tratschin et al., 1986,Mol. Cell. Biol. 6:2884-2894). Due to the inhibitory effects ofexpression of rep on cell growth, constitutive expression of rep in celllines has not been readily achieved. For example, Mendelson et al.(1988, Virology, 166:154-165) reported very low expression of some Repproteins in-certain cell lines after stable integration of AAV genomes.

The capsid proteins VP1, VP2, and VP3 share a common overlappingsequence, but VP1 and VP2 contain additional amino terminal sequences.All three proteins are encoded by the same cap gene reading frametypically expressed from a spliced 2.3 kb mRNA transcribed from the p40promoter. VP2 and VP3 can be generated from this mRNA by use ofalternate initiation codons. Generally, transcription from p40 yields a2.6 kb precursor mRNA which can be spliced at alternative sites to yieldtwo different transcripts of about 2.3 kb. VP2 and VP3 can be encoded byeither transcript (using either of the two initiation sites), whereasVP1 is encoded by only one of the transcripts. VP3 is the major capsidprotein, typically accounting for about 90% of total virion protein. VP1is coded from a minor mRNA using a 3′ donor site that is 30 nucleotidesupstream from the 3′ donor used for the major mRNA that encodes VP2 andVP3. All three proteins are required for effective capsid production.Mutations which eliminate all three proteins (Cap-negative) preventaccumulation of single-strand progeny AAV DNA, whereas mutations in theVP1 amino-terminus (“Lip-negative” or “Inf-negative”) can permitassembly of single-stranded DNA into particles but the infectious titeris greatly reduced.

The genetic analysis of AAV that was highlighted above was largely basedupon mutational analysis of AAV genomes cloned into bacterial plasmids.In early work, molecular clones of infectious genomes of AAV wereconstructed by insertion of double-strand molecules of AAV into plasmidsby procedures such as GC-tailing (Samulski et al., 1982, Proc. Natl.Acad. Sci. USA, 79:2077-2081), addition of synthetic linkers containingrestriction endonuclease cleavage sites (Laughlin et al., 1983, Gene,23:65-73) or by direct, blunt-end ligation (Senapathy & Carter, 1984, J.Biol. Chem., 259:4661-4666). Transfection of such AAV recombinantplasmids into mammalian cells that were also infected with anappropriate helper virus, such as adenovirus, resulted in rescue andexcision of the AAV genome free of any plasmid sequence, replication ofthe rescued genome and generation of progeny infectious AAV particles.This provided the basis for performing genetic analysis of AAV assummarized above and permitted construction of AAV transducing vectors.

Based on the genetic analysis, the general principles of AAV vectorconstruction were defined as reviewed recently (Carter, 1992, CurrentOpinions in Biotechnology, 3:533-539; Muzyczka, 1992, Curr. Topics inMicrobiol. and Immunol., 158:97-129). AAV vectors are generallyconstructed in AAV recombinant plasmids by substituting portions of theAAV coding sequence with foreign DNA to generate a recombinant AAV(rAAV) vector or “pro-vector”. In the vector, the terminal (ITR)portions of the AAV sequence must generally be retained intact becausethese regions are generally required in cis for several functions,including excision from the plasmid after transfection, replication ofthe vector genome and integration and rescue from a host cell genome. Insome situations, providing a single ITR may be sufficient to carry outthe functions normally associated with two wild-type ITRs (see, e.g.,Samulski et al., WO 94/13788, published 23 Jun. 1994).

The vector can then be packaged into an AAV particle to generate an AAVtransducing virus by transfection of the vector into cells that areinfected by an appropriate helper virus such as adenovirus orherpesvirus; provided that, in order to achieve replication andencapsidation of the vector genome into AAV particles, the vector mustgenerally be complemented for any AAV functions required in trans,particularly rep and cap, that were deleted in construction of thevector.

Such AAV vectors are among a small number of recombinant virus vectorsystems which have been shown to have utility as in vivo gene transferagents (reviewed in Carter, 1992, Current Opinion in Biotechnology,3:533-539; Muzyczka, 1992, Curr. Top. Microbiol Immunol. 158:97-129) andthus are potentially of great importance for human gene therapy. AAVvectors are capable of high-frequency transduction and expression in avariety of cells including cystic fibrosis (CF) bronchial and nasalepithelial cells (see, e.g., Flotte et al., 1992a, Am. J. Respir. CellMol. Biol. 7:349-356; Egan et al., 1992, Nature, 358:581-584; Flotte etal., 1993a, J. Biol. Chem. 268:3781-3790; and Flotte et al., 1993b,Proc. Natl. Acad. Sci. USA, 93:10163-10167); human bone marrow-derivederythroleukemia cells (see, e.g., Walsh et al., 1992, Proc. Natl. Acad.Sci. USA, 89:7257-7261); as well as brain, eye and muscle cells. AAV maynot require active cell division for transduction and expression whichwould be another clear advantage over retroviruses, especially intissues such as the human airway epithelium where most cells areterminally differentiated and non-dividing.

There are at least two desirable features of any AAV vector designed foruse in human gene therapy. The first is that the transducing vector begenerated at titers sufficiently high to be practicable as a deliverysystem. This is especially important for gene therapy stratagems aimedat in vivo delivery of the vector. For example, it is likely that formany desirable applications of AAV vectors, such as treatment of cysticfibrosis by direct in vivo delivery to the airway, the desired dose oftransducing vector may be from 10⁸ to 10¹⁰, or, in some cases, in excessof 10¹⁰ particles. Secondly, the vector preparations are preferablyessentially free of wild-type AAV virus (or any replication-competentAAV). The attainment of high titers of AAV vectors has been difficultfor several reasons including preferential encapsidation of wild-typeAAV genomes (if they are present or generated by recombination), and thedifficulty in generating sufficient complementing functions such asthose provided by the wild-type rep and cap genes. Useful cell linesexpressing such complementing functions have been especially difficultto generate, in part because of pleiotropic inhibitory functionsassociated with the rep gene products. Thus, cell lines in which the repgene is integrated and expressed may grow slowly or express rep at verylow levels.

The first AAV vectors described contained foreign reporter genes such asneo, cat or dhfr expressed from AAV transcription promoters or an SV40promoter (Tratschin et al., 1984b, Mol. Cell. Biol. 4:2072-2081;Hermonat & Muzyczka, 1984, Proc. Natl. Acad. Sci. USA, 81:6466-6470;Tratschin et al., 1985, Mol. Cell. Biol. 5:3251-3260; McLaughlin et al.,1988, J. Virol., 62:1963-1973; Lebkowski et al., 1988 Mol. Cell. Biol.,7:349-356). These vectors were packaged into AAV-transducing particlesby co-transfection into adenovirus-infected cells together with a second“packaging plasmid” containing the AAV rep and cap genes expressed fromthe wild-type AAV transcription promoters. Several strategies have beenemployed in attempts to prevent encapsidation of the packaging plasmid.In some cases, (Hermonat & Muzyczka, 1984; McLaughlin et al., 1988) alarge region of bacteriophage lambda DNA was inserted into the packagingplasmid within the AAV sequence to generate an oversized genome thatcould not be packaged. In other cases, (Tratschin et al., 1984b;Tratschin et al., 1985, Lebkowski et al., 1988), the packaging plasmidhad deleted the ITR regions of AAV so that it could not be excised andreplicated and thus could not be packaged. All of these approachesfailed to prevent generation of particles containingreplication-competent AAV DNA and also failed to generate effective hightiters of AAV transducing particles. Indeed, titers of not more than 10⁴infectious particles per ml were cited by Hermonat & Muzyczka, 1984.

In many studies, the presence of overlapping homology between AAVsequences present in the vector and packaging plasmids resulted in theproduction of replication-competent AAV particles. It was shown bySenapathy and Carter (1984, J. Biol. Chem. 259:4661-4666) that thedegree of recombination in such a system is approximately equivalent tothe degree of sequence overlap. It was suggested in a review of theearly work (Carter 1989, Handbook of Parvoviruses, Vol. II, pp. 247-284,CRC Press, Boca Raton, Fla.) that titers of 10⁶ infectious particles perml might be obtained, but this was based on the above-cited studies inwhich large amounts of replication-competent AAV contaminated the vectorpreparation. Such vector preparations containing replication-competentAAV will generally not be preferred for human gene therapy. Furthermore,these early vectors exhibited low transduction efficiencies and did nottransduce more than 1 or 2% of cells in cultures of various human celllines even though the vectors were supplied at multiplicities of up to50,000 particles per cell. This may have reflected in part thecontamination with replication-competent AAV particles and the presenceof the AAV rep gene in the vector. Furthermore, Sanulski et al. (1989,J. Virol. 63:3822-3828) showed that the presence of wild-type AAVsignificantly enhanced the yield of packaged vector. Thus, in packagingsystems where the production of wild-type AAV is eliminated, the yieldof packaged vector may actually be decreased. Nevertheless, for use inany human clinical application it will be preferable to essentiallyeliminate production of replication-competent AAV.

Additional studies (McLaughlin et al., 1988; Lebkowski et al., 1988)attempting to generate AAV vectors lacking the AAV rep or cap genesstill generated replication-competent AAV and still produced very lowtransduction frequencies on human cell lines. Thus, McLaughlin et al.,1988 reported that AAV rep-negative cap-negative vectors containing theneo gene packaged with the same packaging plasmid used earlier byHermonat & Muzyczka (1984) still contained replication-competent AAV. Asa consequence, it was only possible to use this virus at a multiplicityof 0.03 particles per cell (i.e., 300 infectious units per 10,000 cell)to avoid double hits with vector and wild-type particles. Thus, when32,000 cells were infected with 1000 infectious units, an average of 800geneticin-resistant colonies was obtained. Although this was interpretedas demonstrating that the virus was capable of yielding a transductionfrequency of 80%, in fact only 2.5% of the cells were transduced. Thusthe effectively useful titer of this vector was limited. Furthermore,this study did not demonstrate that the actual titer of the vectorpreparation was any higher than those obtained previously by Hermonat &Muzyczka (1984). Similarly, Lebkowski et al., 1988, packaged AAV vectorswhich did not contain either a rep or cap gene, using an ori-negativepackaging plasmid (pBa1A) identical to that used earlier by Tratschin etal., (1984b, 1985), and reported transduction frequencies that weresimilarly low, in that for several human cell lines not more than 1% ofthe cells could be transduced to geneticin resistance even with theirmost concentrated vector stocks. Lebkowski et al., (1988) did not reportthe actual vector titers in a meaningful way but the biological assays,showing not more than 1% transduction frequency when 5×10⁶ cells wereexposed to three ml of vector preparation, indicate that the titer wasless than 2×10⁴ geneticin resistant units per ml. Also, the pBa1Apackaging plasmid contains overlapping homology with the ITR sequence inthe vector and can lead to generation of replication-competent AAV byhomologous recombination.

Laface et al. (1988) used the same vector as that used by Hermonat &Muzyczka (1984) prepared in the same way and obtained a transductionfrequency of 1.5% in murine bone marrow cultures, again showing very lowtiter.

Samulski et al. (1987, J. Virol., 61:3096-3101) constructed a plasmidcalled pSub201 which contained an intact AAV genome in a bacterialplasmid but which had a deletion of 13 nucleotides at the extremity ofeach ITR and thus was rescued and replicated less efficiently than otherAAV plasmids that contained the entire AAV genome. Samulski et al.(1989, J. Virol., 63:3822-3828) constructed AAV vectors based on pSub201but deleted for rep and cap and containing either a hyg or neo geneexpressed from an SV40 early gene promoter. They packaged these vectorsby co-transfection with a packaging plasmid called pAAV/Ad whichconsisted of the entire AAV nucleotide sequence from nucleotide 190 to4490 enclosed at either end with one copy of the adenovirus ITR. In thispackaging plasmid the AAV rep and cap genes were expressed from theirnative AAV promoters (i.e. p5, p19 and p40, as discussed above). Thefunction of the adenovirus ITR in pAAV/Ad was thought to enhance theexpression level of AAV capsid proteins. However, rep is expressed fromits homologous promoter and is negatively regulated and thus itsexpression is limited. Using their encapsidation system, Samulski et al.generated AAV vector stocks that were substantially free ofreplication-competent AAV but had transducing titers of only 3×10⁴hygromycin-resistant units per ml of supernatant. When a wild-type AAVgenome was used in the packaging plasmid, the titer of the AAV vectorprep was increased to 5×10⁴ hygromycin-resistant units per ml. The lowtiter produced in this system thus appears to have been due in part tothe defect in the ITR sequences of the basic pSub201 plasmid used forvector construction and in part due to limiting expression of AAV genesfrom pAAV/Ad. In an attempt to increase the titer of the AAVneo vectorpreparation, Samulski et al. generated vector stocks by transfecting, inbulk, thirty 10-cm dishes of 293 cells and concentrating the vectorstock by banding in CsCl. This produced an AAVneo vector stockcontaining a total of 10⁸ particles as measured by a DNA dot-blothybridization assay. When this vector stock was used at multiplicitiesof up to 1,000 particles per cell, a transduction frequency of 70% wasobtained. This suggests that the particle-to-transducing ratio is about500 to 1,000 particles since at the ratio of one transducing unit percell the expected proportion of cells that should be transduced is 63%according to the Poisson distribution.

Although the system of Samulski et al. (1989), using the vector plasmidpSub201 and the packaging plasmid pAAV/Ad, did not have overlapping AAVsequence homology between the two plasmids, there is overlappinghomology at the XbaI sites and recombination of these sites can lead tothe generation of complete replication-competent AAV. That is, althoughoverlapping homology of AAV sequence is not present, the complete AAVsequence is contained within the two plasmids and the plasmids share ashort (non-AAV) sequence that might facilitate recombination to generatereplication-competent AAV, which is undesirable. That this class ofrecombination occurs in AAV plasmids was shown by Senapathy & Carter(1984, J. Biol. Chem. 259:4661-4666). Given the problems of low titer,and the capability of generating wild-type recombinants, the systemdescribed by Samulski et al., 1989, does not have practical utility forhuman gene therapy.

Several other reports have described AAV vectors. For example,Srivastiva et al., (1989, Proc. Natl. Acad. Sci. USA, 86:8078-8082)described an AAV vector based on the pSub201 plasmid of Samulski et al.(1987), in which the coding sequences of AAV were replaced with thecoding sequences of another parvovirus, B19. This vector was packagedinto AAV particles using the pAAV/Ad packaging plasmid to generate afunctional vector, but titers were not reported. This system was basedon pSub201 and thus suffers from the defect described above for thisplasmid. Second, the vector and the packaging plasmid containedoverlapping AAV sequences (the ITR regions) and thus recombinationyielding contaminating wild-type virus is highly likely.

Chatterjee et al. (1991, Vaccines 91, Cold Spring Harbor LaboratoryPress, pp. 85-89), Wong et al. (1991, Vaccines 91, Cold Spring HarborLaboratory Press, pp. 183-189), and Chatterjee et al. (1992, Science,258:1485-1488) describe AAV vectors designed to express antisense RNAdirected against infectious viruses such as HIV or Herpes simplex virus.However, these authors did not report any titers of their AAV vectorstocks. Furthermore, they packaged their vectors using an ori-negativepackaging plasmid analogous to that used by Tratschin et al. (1984b,1985) containing the Ba1A fragment of the AAV genome and therefore theirpackaging plasmid contained AAV vector sequences that have homology withAAV sequences that were present in their vector constructs. This willalso lead to generation of replication-competent AAV. Thus, Chatterjeeet al., and Wong et al., used a packaging system known to give only lowtiter and which can lead to generation of replication-competent AAVgenomes because of the overlapping homology in the vector and packagingsequences.

Other reports have described the use of AAV vectors to express genes inhuman lymphocytes (Muro-Cacho et al., 1992, J. Immunotherapy,11:231-237) or a human erythroid leukemia cell line (Walsh et al., 1992,Proc. Natl. Acad. Sci. USA, 89:7257-7261) with vectors based on thepSub201 vector plasmid and pAAV/Ad packaging plasmid. Again, titers ofvector stocks were not reported and were apparently low because aselective marker gene was used to identify those cells that had beensuccessfully transduced with the vector.

Transduction of human airway epithelial cells, grown in vitro from acystic fibrosis patient, with an AAV vector expressing the selectivemarker gene neo from the AAV p5 promoter was reported (Flotte et al.,1992, Am. J. Respir. Cell. Mol. Biol. 7:349-356). In this study theAAVneo vector was packaged into AAV particles using the pAAV/Adpackaging plasmid. Up to 70% of the cells in the culture could betransduced to geneticin resistance and the particle-to-transducing ratiowas similar to that reported by Samulski et al. (1989). Thus to obtaintransduction of 70% of the cells, a multiplicity of up to severalhundred vector particles per cell was required. Transduction of humanairway epithelial cells in in vitro culture using an AAV transducingvector that expressed the cystic fibrosis transmembrane conductanceregulator (CFTR) gene from the AAV ITR promoter showed that the cellscould be functionally corrected for the electrophysiological defect inchloride channel function that exists in cells from cystic fibrosispatients (Egan et al., Nature, 1992, 358:581-584; Flotte et al., J.Biol. Chem. 268:3781-3790).

The above-cited studies suggest that AAV vectors have potential utilityas vectors for treatment of human disease by gene therapy. However, thedifficulty in generating sufficient amounts of AAV vectors has been asevere limitation on the development of human gene therapy using AAVvectors. One aspect of this limitation is that there have been very fewstudies using AAV vectors in in vivo animal models (see, e.g., Flotte etal., 1993b; and Kaplitt et al., 1994, Nature Genetics 8:148-154). Thisis generally a reflection of the difficulty associated with generatingsufficient amounts of AAV vector stocks having a high enough titer to beuseful in analyzing in vivo delivery and gene expression.

One of the limiting factors for AAV gene therapy has been the relativeinefficiency of the vector packaging systems that have been used. In theabsence of suitable cell lines expressing sufficient levels of the AAVtrans complementing functions, such as rep and cap, packaging of AAVvectors has been achieved in adenovirus-infected cells byco-transfection of a packaging plasmid and a vector. The efficiency ofthis process is expected to be limited by the efficiency of transfectionof each of the plasmid constructs, and by the low level of expression ofRep proteins from the packaging plasmids described to date. Each ofthese problems appears to relate to the biological activities of the AAVRep proteins which are known to be associated with pleiotropicinhibitory effects. In addition, as noted above, all of the packagingsystems described above have the ability to generatereplication-competent AAV by recombination.

The difficulty in generating cell lines stably expressing functional Repapparently reflects a cytotoxic or cytostatic function of Rep as shownby the inhibition, by Rep protein, of neo-resistant colony formation(Labow et al., 1987; Trempe et al., 1991). This also appears to relateto the tendency of Rep to reverse the immortalized phenotype in culturedcells, which has made the production of cell lines stably expressingfunctional rep extremely difficult. Several attempts to generate celllines expressing rep have been made. Mendelson et al., (1988, Virology,166:154-165) reported obtaining in one cell line some low levelexpression of AAV Rep52 protein but no Rep78 or Rep68 protein afterstable transfection of HeLa or 293 cells with plasmids containing an AAVrep gene. Because of the absence of Rep78 and Rep68 proteins, vectorcould not be produced in the cell line. Another cell line made a barelydetectable amount of Rep78 which was nonfunctional.

Vincent et al. (1990, Vaccines 90, Cold Spring Harbor Laboratory Press,pp. 353-359) attempted to generate cell lines containing the AAV rep andcap genes expressed from the normal AAV promoters, but these attemptswere not successful either because the vectors were contaminated with a100-fold excess of wild-type AAV particles or because the vectors wereproduced at only very low titers of less than 4×10³ infectiousparticles.

Other variations that have been proposed include systems based on theproduction of AAV Cap proteins that might be used to reconstitute AAVparticles, e.g. by assembly in vitro (see, e.g., WO 96/00587, published01 Nov. 1996); systems employing AAV rep-cap genes on a helper virus(see, e.g., WO 95/06743, published 09 Mar. 1995); and systems employinghelper viruses from non-human mammals (see, e.g., WO 95/20671, published03 Aug. 1995).

In yet another approach, Lebkowski et al. (U.S. Pat. No. 5,173,414,issued 22 Dec. 1992) constructed cell lines containing AAV vectors in anepisomal plasmid. These cell lines could then be infected withadenovirus and transfected with the trans-complementing AAV functionsrep and cap to generate preparations of AAV vector. It is claimed thatthis allows higher titers of AAV stocks to be produced. However, in theexamples described, the only information relative to titer that is shownis that one human cell line, K562, could be transduced at efficienciesof only 1% or less, which does not indicate high titer production of anyAAV vector. In this system the vector is carried as an episomal(unintegrated) construct, and it is stated that integrated copies of thevector are not preferred. In a subsequent patent (U.S. Pat. No.5,354,678, issued 11 Oct. 1994), Lebkowski et al. suggest introducingrep and cap genes into the cell genome but the method again requires theuse of episomal AAV transducing vectors comprising an Epstein-Barr virusnuclear antigen (EBNA) gene and an Epstein-Barr virus latent origin ofreplication; and, again, the only information relative to titerindicated that it was fairly low. Similarly, Kotin et al. (WO95/14771,published 01 Jun. 1995) suggested a system employing “first” and“second” vectors to provide a source of an rAAV vector and AAV rep-capgenes, respectively. The proposed system involves a series of sequentialtransfections/infections of the host cells, in a transient transfectionsystem. No data were provided regarding rAAV viral titers obtained and,indeed, it is not apparent that any rAAV virus was actually producedaccording to the suggested system, much less at high titer).

The problem of suboptimal levels of rep expression after plasmidtransfection also relates to another biological activity of theseproteins. There is evidence (Tratschin et al., 1986, Mol. Cell. Biol.6:2884-2894) that AAV Rep proteins down-regulate their own expressionfrom the AAV-p5 promoter which has been used in the various previouslydescribed packaging constructs such as pAAV/Ad (Samulski et al., 1989)or pBa1A (Lebkowski et al., 1988, 1992).

Another attempt to develop cell lines expressing functional rep activitywas recently published by Hölscher et al. (1994, J. Virol.68:7169-7177). They described the generation of cell lines in which repwas placed under control of a glucocorticoid-responsive MMTV promoter.Although they observed particle formation, the particles were apparentlynoninfectious. Additional experiments indicated that the defect wasquite fundamental; namely, there was virtually no accumulation ofsingle-stranded rAAV DNA in the cells. Production of infectiousparticles required an additional transient transfection withconstitutive highly-expressed rep constructs (i.e. they had to “addback” Rep activity to cells that were supposed to be able to provide itthemselves).

There is a significant need for methods that can be used to efficientlygenerate rAAV vectors that are essentially free of wild-type or otherreplication-competent AAV; and a corresponding need for cell lines thatcan be used to effectively generate such rAAV vectors. Several improvedapproaches to generating AAV packaging cell lines have also beendescribed recently, see, e.g., T. Flotte et al., WO 95/13365 (TargetedGenetics Corporation and Johns Hopkins University), and correspondingU.S. Pat. No. 5,658,776; J. Trempe et al., WO 95/13392 (Medical Collegeof Ohio), and corresponding U.S. patent application Ser. No. 08/362,608,now proceeding to issuance as a U.S. patent; and J. Allen, WO 96/17947(Targeted Genetics Corporation). The present invention providesadditional improvements in the production of high-titer rAAV vectorpreparations.

DISCLOSURE OF THE INVENTION

The present invention provides compositions and methods that provideamplifiable expression of the AAV rep and/or cap genes (also referred toherein as “AAV packaging genes”) which can be employed in the generationof recombinant AAV (rAAV) vectors. In particular, the inventors havefound that by removing the AAV rep and/or cap genes from their normalenvironment (i.e. flanked by the AAV ITRs) and placing them inamplifiable linkage with one or more activating elements (exemplified bythe “P1” sequence of human chromosome 19, or analogous elements), it ispossible to obtain controlled but highly amplifiable expression of theAAV packaging genes in cells to be used for the preparation of rAAVvectors. As described and exemplified herein, packaging cassettescomprising rep and/or cap sequences in amplifiable linkage to P1 or aP1-like element can be integrated into the chromosome of a host cell orcan be maintained extrachromosomally as an episome. The methods andcompositions of the present invention can be used to generate stable AAVproducer cells that are capable of supporting production of a very largeburst of rAAV particles upon infection with a suitable helper virus.(such as adenovirus) or provision of helper functions.

Accordingly, in one embodiment, the invention provides a recombinantpolynucleotide sequence encoding an adeno-associated virus (AAV)packaging cassette comprising at least one AAV packaging geneamplifiably linked to a P1 sequence, or an equivalent activatingelement.

In additional embodiments, the invention provides methods for producinghigh-titer stocks of rAAV vectors containing a foreign gene of interest,by co-expressing an rAAV vector containing a gene of interest along withan AAV packaging cassette comprising at least one AAV packaging geneamplifiably linked to an activating element.

The invention also provides compositions and methods for producing celllines comprising an AAV packaging cassette of the invention togetherwith an rAAV vector containing a gene of interest; cell lines producedthereby; compositions and methods for high-efficiency packaging of anrAAV vector containing a gene of interest; and rAAV vectors packagedaccording to the method of the invention.

As illustrated below, AAV packaging cassettes comprising one or moreactivating elements and one or more AAV packaging genes can beintroduced into a host cell and propagated episomally or they can beintegrated into a chromosome of a mammalian host cell. Thus, in anexemplary embodiment, the invention provides AAV packaging cassettescomprising AAV packaging genes and an activating element that arecapable of integrating into the genome of a host cell (such as amammalian cell); as well as packaging cells comprising suchstably-integrated integrated cassettes. In another exemplary embodiment,the invention provides episomal packaging cassettes comprising one ormore AAV packaging genes and one or more activating elements, presentwithin a host cell as a freely-replicating episome (or capable of beingintroduced into a host cell such that, after introduction into the hostcell, the packaging cassette will exist as a freely-replicating episomalelement); as well as packaging cells comprising suchepisomally-maintained packaging cassettes. Illustrative examples of thedesign and use of both types are provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a map of the p5repcapDHFR plasmid.

FIG. 2 shows a map of the P1RCD plasmid.

FIG. 3 shows a map of the episomal packaging plasmid P1/p5repcap/Rep8.The concatameric P1 elements (at “12 o'clock” on the circle) areindicated. Each P1 element comprises a terminal resolution site (TRS)and a Rep-binding site (RBS, also known as a Rep-binding motif or RBMotif). The p5repcap(-P1)/Rep8 construct is identical except that itdoes not contain the concatameric P1 elements.

FIG. 4 shows phosphorimaging analysis of a Southern blot to assay levelsof the episomal P1-containing packaging plasmid in the presence (+) orabsence (−) of Ad5 infection. Lanes 1 and 2-HeLa cells containing theepisomal packaging plasmid p5repcap(-P1)/Rep8. Lanes 3 and 4, 5 and6-HeLa cells containing the episomal packaging plasmid P1/p5repcap/Rep8.

FIG. 5 shows phosphorimaging analysis of a Southern blot to assayrAAV-CFTR production in cells containing the episomal packaging plasmidsp5repcap(-P1)/Rep8 and P1 /p5repcap/Rep8, in the presence (+) or absence(−) of Ad5 infection. Lanes 1 and 2-HeLa cells containing the episomalpackaging plasmid p5repcap(-P1)/Rep8. Lanes 3 and 4, 5 and 6-HeLa cellscontaining the episomal packaging plasmid P1/p5repcap/Rep8.

DETAILED DESCRIPTION OF THE INVENTION

A basic challenge in the area of gene therapy is the development ofstrategies for efficient gene delivery to cells and tissues in vivo. Onestrategy involves the use of adeno-associated virus (AAV) vectors.Recombinant AAV vectors are recombinant constructs of the AAV genomecomprising sequences required in cis for vector packaging (typically AAVITR sequences), along with heterologous polynucleotide(s) encoding aprotein or function of interest. Recombinant AAV vectors are potentiallypowerful tools for human gene therapy.

Although rAAV vectors are capable of in vivo gene delivery, for examplein the respiratory tract, high titers of such vectors are necessary toallow the delivery of a sufficiently high multiplicity of vector in aminimal volume. Consequently, optimal packaging methodology is ofcentral importance for AAV-mediated gene therapy approaches. Packagingof rAAV vectors is mediated by the products of two AAV genes: rep(replication proteins) and cap (capsid proteins), which can be providedseparately in trans. A sequence comprising AAV packaging genes to beprovided in trans is often referred to herein as a “packaging cassette”.It is thus desirable to construct packaging cell lines containing boththe AAV packaging genes (e.g., in a packaging cassette) and an rAAVvector. However, stable, helper-free AAV packaging cell lines have beendifficult to obtain, primarily due to the activities of Rep and Capproteins, for which low-level expression can impose a severe constrainton packaging, while high-level expression (particularly of Rep proteins)can negatively affect the host cell (see Background). The presentinvention provides controlled but amplifiable expression of the rep andcap genes, to thereby provide Rep and capsid proteins at levelssufficient for production of high-titer vector stocks, while avoidingany effects of cell toxicity (as can occur if the rep gene is placedunder the control of regulatory elements that exhibit some constitutiveactivity or are not tightly regulated).

The methods and compositions of the present invention, which allow forcontrolled, amplifiable expression of AAV packaging genes, even when thepackaging genes are expressed from their native promoters (such as therep gene p5 promoter, which is a relatively weak promoter), providesubstantial improvements in packaging efficiency. This is accomplishedby providing AAV packaging genes in a recombinant DNA construct whereinthey are amplifiably linked to an activating element. In preferredembodiments, the activating element is directly or indirectly triggeredby the user when it is desired to initiate vector production, preferablyby infection with helper virus or provision of helper function. The useof the P1 sequence of human chromosome 19 is exemplary in theserespects. Thus, in the absence of adenovirus infection (or equivalenthelper function), little if any Rep is produced from the p5 promoter,which is relatively weak or inactive in the absence of helper virusinfection or provision of helper function (e.g., adenovirus infection orinclusion in the host cells of helper functions, such as E1A activity inhuman 293 cells). Without wishing to be bound by theory, it appears thatupon infection or provision of helper function, the p5 promoter isturned on to some degree, resulting in the synthesis of some Repprotein, which may then, by acting via the P1 activating element,trigger an amplification event by which the linked rep and/or cap genesare amplified—thereby serving as the basis for a much higher level ofexpression. The activating element, exemplified by P1, can thus promoteamplification of AAV packaging genes to which it is linked. Theresulting elevation in template levels would allow the gene products(like Rep and Cap proteins) to be produced in much higher amounts,particularly in view of the fact that their promoters can also betranscriptionally activated to thereby provide efficient packagingfunctions. Inclusion of an activating element in an AAV packagingcassette, along with AAV packaging genes, thus provides a new type ofAAV packaging cassette which is particularly useful in the production ofhigh-titer stocks of rAAV vectors, as described and exemplified herein.

Some previous attempts to incorporate rep genes into a host cell mayhave resulted in either of two undesirable alternatives: (1) host cellscontaining a stably-integrated, expressed rep gene in which cytotoxicand/or cytostatic effects limit cell growth and/or led to poor titers ofrAAV vectors; or (2) host cells exhibiting normal growth rates, butnevertheless having little capability for generating high titers of rAAVvectors (possibly reflecting integration at transcriptionally silentsites, sequence rearrangements, etc.). Without wishing to be bound bytheory, it is proposed that the AAV packaging cassettes of the presentinvention can also be used to effectively provide a baseline level ofRep proteins that is very low (if present at all) and is therefore notdetrimental to the growth of the host cell, but can be amplified whenrequired (for example by helper virus infection or provision of helperfunction) to a level that promotes efficient production of rAAV vectors.Thus, when brought about under the control of the user, amplificationresults in increased levels of templates comprising AAV packaging genes,which collectively allow high levels of expression of packaging geneproducts (e.g., Rep and Cap proteins), which in turn facilitatesproduction of high titers of rAAV genomes.

In the case of the wild-type AAV, for example, it is generally believedthat the native promoter for Rep protein expression (p5) is relativelyweak and consequently that synthesis of native Rep proteins does notoccur to any substantial degree in the absence of stimulatory factorssuch as the E1A proteins provided by adenovirus as a helper virus, orequivalent helper functions. (It should be noted that human 293 cellscontain portions of the human adenovirus genome, in particular the E1region, that appear to stimulate the p5 promoter.) In addition to therelatively low activity of the rep p5 promoter in the absence of helperfunction, it appears that AAV Rep proteins can effectively modulatetheir own expression. Both of these phenomena tend to preventreplication from occurring when the virus is in the latent proviralstate.

The present invention effectively provides for controlled amplificationof DNA comprising the packaging cassettes of the invention, therebyproviding increased template levels for synthesis of AAV packagingproteins. Thus, in the packaging cassettes of the invention, AAVpackaging genes can be operably linked to relatively weak promoters andnevertheless be capable of providing acceptable levels of packagingproteins upon activation. In preferred embodiments, AAV packaging genesare operably linked to their native promoters (i.e., p5, p19 and p40, inthe case of AAV2 as described above). Since p5 is an extremely weakpromoter, and virtually no transcription initiated from p5 is observedin the absence of helper function, an AAV packaging cassette whereinpackaging gene expression is controlled by p5 is not likely to have anyRep-dependent cytostatic effect on the host cell prior to activation andamplification. However, upon activation by helper virus infection orprovision of helper function, the packaging cassette template isamplified, leading to a greater number of templates for transcription ofAAV packaging proteins. Furthermore, helper virus infection or provisionof helper function is believed also to stimulate transcription from thep5 promoter (which regulates synthesis of mRNA encoding Rep proteins).Accordingly, in the packaging cassettes of the invention, expression ofAAV packaging genes is preferably not triggered until provision ofhelper function (i.e., at the time the host cells are to be used forpackaging of rAAV particles), thereby avoiding the accumulation of high(and potentially cytostatic or cytotoxic) levels of AAV packagingproteins prior to the time they are required for packaging. Thesepreferred embodiments thus provide two levels of augmentation ofpackaging protein synthesis, in which a helper function-dependentactivating element is amplifiably linked to sequences encoding AAVpackaging genes, whose promoters are also stimulated by helper function.

The inventors have shown, as described below, that AAV packagingcassettes comprising activating elements (as exemplified by the P1sequence element) can be used to generate dramatic increases in thelevels of vector production. The use of P1 sequences as activatingelements for the AAV packaging cassettes of the present invention isbelieved to be particularly convenient since the same event that isrequired to trigger the productive generation of AAV particles (i.e.provision of helper virus or helper functions) is believed to alsotrigger amplification of a construct containing an activating elementsuch as P1 (such as, for example, a packaging cassette of theinvention), and up-regulate the AAV promoters (including P5), resultingin both provision of increased template and in higher levels ofsynthesis of the packaging gene products (i.e. AAV packaging proteins)from the amplified templates. Thus, according to the present invention,the coupling of activating elements, such as P1, with AAV packaginggenes can provide a combination of advantages including control ofpackaging gene product levels (in the “pre-activated” state) and, uponactivation, amplification of template levels and stimulation oftranscription.

It is also noted that, in many cases, the activation of replicationorigins is, or can be, subject to strict control. Accordingly, variousreplication origins, such as those present in eukaryotic or prokaryoticchromosomes, viral genomes, organelle genomes, and bacteriophagegenomes, for example, and other origin-like or “ori-like” sequences canbe used in the practice of the invention (e.g., as alternatives oradditions to the use of P1). Such “activatable” origins are those thatare not constitutive, but rather require a signal before replicationinitiation and subsequent amplification of linked sequences will occur.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual,Second Edition (1989); Oligonucleotide Synthesis (M. J. Gait Ed., 1984);Animal Cell Culture (R. I. Freshney, Ed., 1987); the series Methods inEnzymology (Academic Press, Inc.); Gene Transfer Vectors for MammalianCells (J. M. Miller and M. P. Calos eds. 1987); Handbook of ExperimentalImmunology, (D. M. Weir and C. C. Blackwell, Eds.); Current Protocols inMolecular Biology (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore,J. G. Seidman, J. A. Smith, and K. Struhl, eds., 1987); and CurrentProtocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach and W. Strober, eds., 1991).

All patents, patent applications, and publications mentioned herein,both supra and infra, are hereby incorporated herein by reference.

Definitions

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably to refer to polymers of amino acids of any length. Theseterms also include proteins that are post-translationally modifiedthrough reactions that include, but are not limited to, glycosylation,acetylation and phosphorylation.

“Polynucleotide” refers to a polymeric form of nucleotides of anylength, either ribonucleotides or deoxyribonucleotides, or analogsthereof. This term refers only to the primary structure of the molecule.Thus, double- and single-stranded DNA, as well as double- andsingle-stranded RNA are included. It also includes modifiedpolynucleotides such as methylated or capped polynucleotides.

A “gene” refers to a polynucleotide containing at least one open readingframe that is capable of encoding a particular protein after beingtranscribed and translated.

A “transcriptional regulatory sequence” as used herein, refers to anucleotide sequence that controls the transcription of a gene or codingsequence to which it is operably linked. Transcriptional regulatorysequences of use in the present invention generally include at least onetranscriptional promoter and may also include one or more enhancersand/or terminators of transcription.

A “promoter,” as used herein, refers to a nucleotide sequence thatdirects the transcription of a gene or coding sequence to which it isoperably linked.

“Operably linked” refers to an arrangement of two or more components,wherein the components so described are in a relationship permittingthem to function in a coordinated manner. By way of illustration, atranscriptional regulatory sequence or a promoter is operably linked toa coding sequence if the transcriptional regulatory sequence or promoterpromotes transcription of the coding sequence. An operably linkedtranscriptional regulatory sequence is generally joined in cis with thecoding sequence, but it is not necessarily directly adjacent to it.

“Recombinant,” refers to a genetic entity distinct from that generallyfound in nature. As applied to a polynucleotide or gene, this means thatthe polynucleotide is the product of various combinations of cloning,restriction and/or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is compared. For example, apolynucleotide introduced by genetic engineering techniques into adifferent cell type is a heterologous polynucleotide (and, whenexpressed, can encode a heterologous polypeptide). Similarly, atranscriptional regulatory sequence or promoter that is removed from itsnative coding sequence and operably linked to a different codingsequence is a heterologous transcriptional regulatory sequence orpromoter.

A “vector”, as used herein, refers to a recombinant plasmid or virusthat comprises a polynucleotide to be delivered into a host cell, eitherin vitro or in vivo. The polynucleotide to be delivered, sometimesreferred to as a “target polynucleotide,” “transgene”, or “gene ofinterest” may comprise a coding sequence of interest in gene therapy(such as a gene encoding a protein of therapeutic interest) and/or aselectable or detectable marker.

A “replicon” refers to a polynucleotide comprising an origin ofreplication which allows for replication of the polynucleotide in anappropriate host cell. Examples of replicons include episomes (includingplasmids), as well as chromosomes (such as the nuclear or mitochondrialchromosomes).

An “origin,” “replication origin,” “ori-like sequence” or “ori element”is a nucleotide sequence involved in one or more aspects of initiationof DNA replication, such as, for example, binding of replicationinitiation factors, unwinding of the DNA duplex, primer formation,and/or template-directed synthesis of a complementary strand. Asdiscussed in detail herein and in the art, ori-like sequences cangenerally be found in any polynucleotide that is naturally replicated,including plasmids and viruses, as well as prokaryotic, mitochondrialand chloroplast genomes and eukaryotic chromosomes. Such ori-likesequences can be identified genetically (i.e., replication-defectivemutants, ars sequences) or functionally (i.e., through biochemicalassay, electron microscopy, etc.), as is known in the art.

“Stable integration” of a polynucleotide into a cell means that thepolynucleotide has been integrated into a replicon that tends to bestably maintained in the cell. Although episomes such as plasmids cansometimes be maintained for many generations, genetic material carriedepisomally is generally more susceptible to loss thanchromosomally-integrated material. However, maintenance of apolynucleotide can often be effected by incorporating a selectablemarker into or adjacent to a polynucleotide, and then maintaining cellscarrying the polynucleotide under selective pressure. In some cases,sequences cannot be effectively maintained stably unless they havebecome integrated into a chromosome; and, therefore, selection forretention of a sequence comprising a selectable marker can result in theselection of cells in which the marker has become stably-integrated intoa chromosome. Antibiotic resistance genes can be conveniently employedas such selectable markers, as is well known in the art. Typically,stably-integrated polynucleotides would be expected to be maintained onaverage for at least about twenty generations, preferably at least aboutone hundred generations, still more preferably they would be maintainedpermanently. The chromatin structure of eukaryotic chromosomes can alsoinfluence the level of expression of an integrated polynucleotide.Having the genes carried on stably-maintained episomes can beparticularly useful where it is desired to have multiplestably-maintained copies of a particular gene. The selection of stablecell lines having properties that are particularly desirable in thecontext of the present invention are described and illustrated below.

“AAV” is adeno-associated virus. Adeno-associated virus is a defectiveparvovirus that grows only in cells in which certain functions areprovided by a co-infecting helper virus. General reviews of AAV may befound in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. I,pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (NewYork). The AAV2 serotype was used in some of the illustrations of thepresent invention that are set forth in the Examples. However, it isfully expected that these same principles will be applicable to otherAAV serotypes since it is now known that the various serotypes are quiteclosely related—both functionally and structurally, even at the geneticlevel (see, e.g., Blacklow, 1988, pp. 165-174 of Parvoviruses and HumanDisease, J. R. Pattison (ed.); and Rose, 1974, Comprehensive Virology 3:1-61). For example, all AAV serotypes apparently exhibit very similarreplication properties mediated by homologous rep genes; and all bearthree related capsid proteins such as those expressed in AAV2. Thedegree of relatedness is further suggested by heteroduplex analysiswhich reveals extensive cross-hybridization between serotypes along thelength of the genome; and the presence of analogous self-annealingsegments at the termini that correspond to inverted terminal repeats(ITRs). The similar infectivity patterns also suggest that thereplication functions in each serotype are under similar regulatorycontrol.

A “recombinant AAV vector” (or “rAAV vector”) refers to a vectorcomprising one or more polynucleotide sequences of interest, genes ofinterest or “transgenes” that are flanked by AAV inverted terminalrepeat sequences (ITRs). Such rAAV vectors can be replicated andpackaged into infectious viral particles when present in a host cellthat has been infected with a suitable helper virus and that isexpressing AAV rep and cap gene products (i.e. AAV Rep and Capproteins). When an rAAV vector is incorporated into a largerpolynucleotide (e.g in a chromosome or in another vector such as aplasmid used for cloning or transfection), then the rAAV vector istypically referred to as a “pro-vector” which can be “rescued” byreplication and encapsidation in the presence of AAV packaging functionsand necessary helper functions.

A “helper virus” for AAV refers to a virus that allows AAV (which is a“defective” parvovirus) to be replicated and packaged by a host cell. Anumber of such helper viruses have been identified, includingadenoviruses, herpesviruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C (Ad5) is most commonly used. Numerousadenoviruses of human, non-human mammalian and avian origin are knownand available from depositories such as the ATCC. Viruses of the herpesfamily include, for example, herpes simplex viruses (HSV) andEpstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) andpseudorabies viruses (PRV); which are also available from depositoriessuch as ATCC. “Helper function” refers to the activity provided by thehelper virus that allows replication and packaging of an AAV genome, orany equivalent activity. Helper functions are also believed to stimulatetranscription of some AAV promoters, including p5, and may enhanceprocessivity of replication in cells in which helper functions areexpressed.

“Packaging” as used herein refers to a series of subcellular events thatresults in the assembly and encapsidation of a viral vector,particularly an rAAV vector. Thus, when a suitable vector is introducedinto a packaging cell line under appropriate conditions, it can beassembled into a viral particle. Functions associated with packaging ofviral vectors, particularly rAAV vectors, are described herein and inthe art.

AAV “rep” and “cap” genes are genes encoding replication andencapsidation proteins, respectively. AAV rep and cap genes have beenfound in all AAV serotypes examined, and are described herein and in thereferences cited. In wild-type AAV, the rep and cap genes are generallyfound adjacent to each other in the viral genome (i.e. they are“coupled” together as adjoining or overlapping transcriptional units),and they are generally conserved among AAV serotypes. AAV rep and capgenes are also individually and collectively referred to herein as “AAVpackaging genes.” AAV packaging genes that have been modified bydeletion or point mutation, or which have been subdivided intocomponents which can be rejoined by recombination (e.g., as described inco-owned U.S. patent application Ser. No. 60/041,609, filed 18 Dec.1996, the disclosure of which is hereby incorporated by reference), mayalso be used in the present invention. AAV packaging genes can also beoperably linked to other transcriptional regulatory sequences, includingpromoters, enhancers and polyaderiylation (“polyA”) sequences (whichadditional transcriptional regulatory sequences can also beheterologous). An “AAV packaging cassette” is a recombinant constructwhich includes one or more AAV packaging genes.

“Efficiency” when used in describing a cell line refers to certainuseful attributes of the line; in particular, the growth rate, and (forpackaging cell lines) the number of virus particles produced per cell.“Efficient growth” of a packaging cell line refers to the effectivegrowth rate of the packaging cell, related to a comparable parental-typecell (i.e., a cell that does not carry an introduced AAV packaging gene)Preferably, the relative growth rate is at least 20% of the parentaltype, more preferably, 40%, more preferably, 80%, still more preferably,90% and, most preferably, 100%. “High efficiency packaging” indicatesproduction of at least about 100 viral particles per cell, morepreferably at least about 1,000 viral particles per cell, still morepreferably at least about 10,000 viral particles per cell. “High safetypackaging” indicates that, of the recombinant AAV viral particlesproduced, fewer than about 1 in 10⁶ are replication-competent AAV viralparticles, preferably fewer than about 1 in 10⁸ arereplication-competent, more preferably fewer than about 1 in 10¹⁰ arereplication-competent, still more preferably fewer than about 1 in 10¹²are replication-competent, most preferably none arereplication-competent. Preferred packaging cells of the presentinvention exhibit combinations of such high efficiency and high safety.

“Host cells”, “cell lines”, “cell cultures”, “packaging cell line” andother such terms denote higher eukaryotic cells, preferably mammaliancells, most preferably human cells, useful in the present invention.These cells can be used as recipients for recombinant vectors, virusesor other transfer polynucleotides, and include the progeny of theoriginal cell that was transduced. It is understood that the progeny ofa single cell may not necessarily be completely identical (in morphologyor in genomic complement) to the original parent cell.

A “therapeutic gene”, “target polynucleotide”, “transgene”, “gene ofinterest” and the like generally refer to a gene or genes to betransferred using a vector. Typically, in the context of the presentinvention, such genes are located within the rAAV vector (which vectoris flanked by inverted terminal repeat (ITR) regions and thus can bereplicated and encapsidated into rAAV particles). Target polynucleotidescan be used in this invention to generate rAAV vectors for a number ofdifferent applications. Such polynucleotides include, but are notlimited to: (i) polynucleotides encoding proteins useful in other formsof gene therapy to relieve deficiencies caused by missing, defective orsub-optimal levels of a structural protein or enzyme; (ii)polynucleotides that are transcribed into anti-sense molecules; (iii)polynucleotides that are transcribed into decoys that bind transcriptionor translation factors; (iv) polynucleotides that encode cellularmodulators such as cytokines; (v) polynucleotides that can makerecipient cells susceptible to specific drugs, such as the herpes virusthymidine kinase gene; (vi) polynucleotides for cancer therapy, such asE1A tumor suppressor genes or p53 tumor suppressor genes for thetreatment of various cancers and (vii) polynucleotides that encodeantigens or antibodies. To effect expression of the transgene in arecipient host cell, it is preferably operably linked to a promoter orother such transcriptional regulatory sequence, either its own or aheterologous promoter. A large number of suitable promoters are known inthe art, the choice of which depends on the desired level of expressionof the target polynucleotide; whether one wants constitutive expression,inducible expression, cell-specific or tissue-specific expression, etc.The rAAV vector may also contain a selectable marker.

An “activating element” is a sequence that responds to the presence ofan activation signal by amplifying (i.e., replicating the sequences) towhich it is amplifiably linked. A preferred activating element is the P1element and preferred activation signals include AAV helper functions(as exemplified by adenovirus E1A function) or their equivalents. Asused herein, two sequences, one of which is an activating element, are“amplifiably linked” when they are in sufficient proximity to each otherthat replication initiating from the activating element results inamplification (i.e., increased copy number) of the other sequence.Preferably, the copy number of the amplified sequence is amplified2-fold or greater, more preferably, 10-fold or greater, still morepreferably, 20-fold or greater. It is to be noted that the ability of anactivating element to amplify an amplifiably-linked sequence will beinfluenced by the degree of processivity of replication initiating fromthe activating element. Thus, factors that enhance processivity ofreplication will tend to increase the effective level of amplificationof a sequence that is amplifiably linked to an activating element. Inthe context of the present invention, infection with adenovirus, orprovision of equivalent helper function, may enhance processivity ofreplication as well as initiating amplification.

Sequences Activating Amplification and Controlled, High-EfficiencyExpression of AAV Packaging Genes.

The present inventors have discovered that activating elements such asthe P1 sequence (normally found on human chromosome 19), whenamplifiably linked to AAV packaging genes, can provide controlled,amplifiable expression of the linked packaging genes and/or a dramaticincrease in the ability of such genes to support the production of hightiters of rAAV vectors. In particular, when an AAV packaging cassette ofthe present invention is co-expressed in host cells with an rAAV vector(containing one or more genes of interest flanked by AAV ITR sequences)under suitable conditions including the provision of helper virus orhelper function, high titers of AAV virus containing the rAAV vector areproduced by the host cells. Thus, P1 exemplifies a class of activatingelements possessing, among other properties, activatable replicationfunction, that is useful in the construction of AAV packaging cassettesto promote production of high-titer stocks of rAAV vectors.

The methods and compositions of the invention will therefore utilizerecombinant DNA constructs wherein AAV packaging genes are amplifiablylinked to one or more activating elements. The presently preferredactivating elements are exemplified by P1 and P1-like elements thatexhibit structural and functional properties related to initiation ofreplication. Most preferred are elements that act as helperfunction-inducible origins of replication. Other sequences that can bedirectly or indirectly induced to initiate replication in response tohelper function will also be useful in the practice of the invention. Inaddition, sequences that are not inducible by helper function, but whichcan be induced to initiate DNA replication by other stimuli (providedand/or controlled by the user), are also useful as activating elementsin the practice of the invention. Examples of such other inducibleactivating elements would include, by way of illustration, a sequence atwhich replication is initiated in the presence of a replication proteinthat is itself inducible (e.g., a temperature-sensitive replicationprotein that can be activated by a shift to permissive temperature, or areplication protein whose gene is placed under the control of aninducible promoter). Naturally-occurring activating elements having thedesired properties can be isolated; alternatively, synthetic sequencescan be designed based, in whole or in part, on the observedrelationships between structure and function found innaturally-occurring activating elements.

The P1 element contains at least two distinct sequence motifs, a site atwhich Rep proteins can bind, known as the “Rep-binding motif” (or“Rep-binding site”) and a terminal resolution site, at which bound Repprotein can nick the DNA (see Example A 1). During AAV replication, itis believed that Rep protein binds within the AAV inverted terminalrepeat and catalyzes the formation of a nick (at the terminal resolutionsite), resulting in covalent attachment of Rep protein to the newlygenerated 5′ end. The 3′ end of the nick serves as a primer for AAV DNAsynthesis. Consequently, the Rep binding motif and/or the terminalresolution sequence, alone or in combination, may form all or part of anactivating element for expression of AAV packaging genes. Furthermore,binding and cleavage of a sequence by Rep proteins can be used as anassay to identify additional activating elements.

With respect to the use of inducible origins as activating elements, itis noted that origin sequences in eukaryotes (“ori sequences”) aregenerally associated with several characteristic functions including,but not limited to, protein binding, DNA unwinding and template-directedchain elongation. See, for example, Kornberg and Baker (1992) DNAREPLICATION, Second Edition, W. H. Freeman & Co., New York; Boulikas(1996) J. Cell Biochem. 60:297-316; and Diffley (1996) Genes & Devel.10:2819-2830. Accordingly, sequences having one or any combination ofthese properties can find use as activating elements in the practice ofthe present invention.

For instance, various initiator proteins bind at or near the orisequence to facilitate initiation of DNA replication. Accordingly,sequences capable of binding such initiator proteins, and the initiatorproteins themselves (and their encoding genes) can find use in thepractice of the invention. Determination of the ability of a particularprotein to bind to an ori sequence can be assayed by several methodsthat are well-known in the art, including, but not limited to,sedimentation, nuclease protection, filter binding, gel mobility-shift,and various affinity techniques, including, but not limited to, DNAaffinity matrices. Assays for origin function are well-known in the artand include electron microscopy, genetic analysis and template-directedincorporation of labeled nucleoside triphosphate, to name just a few.Activation of origin function can be detected as an increase in level ofreplication as determined by the above-mentioned origin assays. Thus,origin sequences can be identified, proteins that interact with aparticular origin can also be identified, and the ability of anori-binding protein to activate a particular origin can be determined bymethods that are well-known in the art.

Thus, in additional embodiments, activating elements can take the formof inducible replication origins, such as mammalian, viral,mitochondrial, chloroplast, plasmid or bacteriophage replicationorigins, for example.

Accordingly, an inducible origin can be amplifiably linked to AAVpackaging genes in an AAV packaging cassette and the packaging cassettecan be introduced into suitable host cells containing an rAAV vector (orto which an rAAV vector is added simultaneously or subsequently). Whenpackaging of the rAAV vector is required, the host cells are providedwith a molecule, such as a protein, which activates the inducibleorigin, along with a helper function. The activating molecule can beprovided directly. Alternatively, if the activating molecule is aprotein, then a gene (or genes) encoding the protein, under thetranscriptional control of an inducible promoter, can be present in thehost cells. In this case stimulation of transcription of the gene(s)encoding the activating protein can be achieved by provision of theappropriate inducing molecule, or the gene(s) encoding the activatingprotein can be placed under the control of a promoter that is activatedby a helper function, such as adenovirus infection. An appealing featureof the latter method is that the same signal (i.e., provision of helperfunction) can be responsible for transcriptional stimulation of both thegene(s) encoding the inducing molecule(s) and AAV packaging genes(since, for instance, transcription from the p5 promoter is thought tobe stimulated by helper function, such as adenovirus infection). Foradditional, non-limiting examples of promoters that are inducible byhelper function (and methods to identify such promoters) see, forexample, co-owned PCT Publication WO 96/17947, the disclosure of whichis hereby incorporated by reference in its entirety. Further examples ofinducible promoters include, but are not limited to, the MMTV LTRpromoter, which is inducible by glucocorticoids, and the metallothioneinpromoter, which is inducible by heavy metals. Many other induciblepromoters are known in the art and can be used in this aspect of theinvention.

Among other sequence that are commonly associated with origins ofreplication are palindromic sequences, sequences having the potential toform cruciform structures, DNA unwinding elements, sequences involved insynthesis or recruitment of replication primers, bent or curved DNA(which can be detected by its altered electrophoretic mobility),nuclease sensitive sequences, and nuclear matrix attachment sites. See,for example, Boulikas (1996) J. Cell Biochem. 60:297-316; and Diffley(1996) Genes & Devel. 10:2819-2830. In addition, sequences involved inchromosomal or extrachromosomal gene amplification can also be used asactivating elements. To provide just one example, amplification of thedihydrofolate reductase (DHFR) gene occurs in response to methotrexate.

Sequences possessing origin activity, which may be useful as activatingelements, can also be identified by electron microscopic analysis ofreplicating DNA molecules. See, for example, Fareed et al. (1980) Meth.Enzymology, vol. 65 (eds. L. Grossman and K. Moldave), Academic Press,New York, pp. 709-717.

Assays for ori-like sequences that can serve as activating elements inthe present invention have been described above and are well-known tothose of skill in the art. In addition, proteins, such as Rep, whichinteract with particular activating elements, can be identified bymethods well-known in the art, including those described above, and usedfor the identification of additional activating elements.

Orientation and Spacing of Activating Elements with Respect to AAVPackaging Genes

We have observed that placing an activating element, as exemplified by aP1 sequence, near to a cassette comprising AAV packaging genes resultedin a dramatic increase in the ability of the packaging genes to supportthe production of recombinant AAV vectors. Indeed, as shown below, a P1element placed more than 4 kb downstream of the rep gene transcriptionalstart site in an integrated AAV packaging cassette resulted inapproximately a 14-fold amplification of the packaging cassette (seeExample A 11 below) and close to a 1,000-fold increase in rAAV virustiter (see Example A 12 below), compared to cells containing a packagingcassette lacking a P1 element. Although placing an activating elementfurther away from an AAV packaging gene (e.g. 5-10 kb or further) mayresult in somewhat lower activity, longer distances between anactivating element and its amplifiably-linked AAV packaging genes wouldstill be expected to provide a degree of activation sufficient forimproved rAAV production, especially under conditions in whichprocessivity of replication is enhanced, as discussed above. Where P1 isused as an activating element, it can be desirable to have at least somespacer sequence (e.g. about 0.5 to 1 kb) between the P1 sequence and theAAV packaging genes in order to reduce or eliminate the possibility thatrecombination between P1 and an ITR sequence could regenerate areplication-competent AAV genome that would be of a size that could beefficiently packaged.

In amplifying copies of integrated AAV in response to helper virusinfection, the P1 element appears to direct amplificationunidirectionally. Without wishing to be bound by theory, it is believedthat interaction of Rep with a Rep-binding motif may be followed bynicking between the two T residues in a Terminal Resolution Site (TRS),as illustrated below. Subsequently, replication may initiate from the 3′hydroxyl end of the nick and proceed toward the Rep-binding motif.Accordingly, it is presently preferred that a unidirectional activatingelement as in the case of P1 be oriented such that unidirectionalreplication proceeds from the activating element toward the associatedAAV packaging gene(s). Alternatively, AAV packaging genes can be flankedby activating elements that are oriented so that replication initiatedat each element proceeds “inward” toward the AAV packaging gene(s).However, bidirectional activating elements are also useful in thepractice of the invention, since, in these cases, one of the twodirections of replication will proceed toward the associated AAVpackaging genes. Furthermore, for episomal packaging cassettes, aunidirectional activating element wherein replication is oriented awayfrom associated AAV packaging genes can also be useful, sincereplication will proceed around the circular episomal genome andeventually encounter the associated AAV packaging gene sequences. Onecan also incorporate multiple copies of such activating elements, whichcan be oriented to promote replication in both directions. Exemplaryillustrations of such constructs are provided below.

Addition of multiple activating elements to an AAV packaging cassettewould be expected to provide further degrees of amplification. Forexample, two P1 elements that are oriented such that replicationinitiated from each progresses in opposite directions would providecorrespondingly higher levels of amplification of linked sequences.Thus, insertion of a second P1 element into a construct such as P1RCD insuch an orientation as to amplify the opposite strand of an integratedpackaging construct should increase amplification, Rep and Cap levelsand rAAV virus production.

In general, addition of multiple activating elements to the AAVpackaging cassettes of the invention should increase amplification andtherefore should increase levels of AAV packaging gene products.Consequently, production of rAAV vectors and virus production shouldalso be increased under these conditions, compared to situations inwhich a single activating element is present in a packaging cassette.

Production of RAAV Vectors

To generate recombinant AAV particles useful for such purposes as genetherapy, the packaging cell line is generally supplied with arecombinant AAV vector comprising AAV inverted terminal repeat (ITR)regions surrounding one or more polynucleotides of interest (or “target”polynucleotides).

The target polynucleotide, if it is intended to be expressed, isgenerally operably linked to a promoter, either its own or aheterologous promoter. A large number of suitable promoters are known inthe art, the choice of which depends on the desired level of expressionof the target polynucleotide; whether one wants constitutive expression,inducible expression, cell-specific or tissue-specific expression, etc.The rAAV vector can also contain a positive selectable marker in orderto allow for selection of cells that have been infected by the rAAVvector; and/or a negative selectable marker (as a means of selectingagainst those same cells should that become necessary or desirable);see, e.g., S. D. Lupton, PCT/US91/08442 and PCT/US94/05601.

By way of illustration, we have used rAAV vectors containingpolynucleotides that encode a functional cystic fibrosis transmembraneconductance regulator polypeptide (CFTR) operably linked to a promoter.As is now known in the art, there are a variety of CFTR polypeptidesthat are capable of reconstituting CFTR activity in cells derived fromcystic fibrosis patients. For example, Carter et al. have describedtruncated variants of CFTR genes that encode functional CFTR proteins(see, e.g., U.S. Ser. No. 08/455,552, filed 31 May 1995, now proceedingto issuance). See also, Rich et al. (1991, Science, 253: 205-207) whohave described a CFTR derivative missing amino acid residues 708-835,that was capable of transporting chloride and capable of correcting anaturally occurring CFTR defect, and Egan et al. (1993) who describedanother CFTR derivative (comprising about 25 amino acids from anunrelated protein followed by the sequence of native CFTR beginning atresidue 119) that was also capable of restoring electrophysiologicalcharacteristics of normal CFTR. To take two additional examples, Arispeet al. (1992, Proc. Natl. Acad. Sci. USA 89: 1539-1543) showed that aCFTR fragment comprising residues 433-586 was sufficient to reconstitutea correct chloride channel in lipid bilayers; and Sheppard et al. (1994,Cell 76: 1091-1098) showed that a CFTR polypeptide truncated at residue836 to about half its length was still capable of building a regulatedchloride channel. Thus, the native CFTR protein, and mutants andfragments thereof, all constitute CFTR polypeptides that are useful inthe practice of this invention.

Other useful target polynucleotides can be used in this invention togenerate rAAV vectors for a number of different applications. Suchpolynucleotides include, but are not limited to: (i) polynucleotidesencoding proteins useful in other forms of gene therapy to relievedeficiencies caused by missing, defective or sub-optimal levels of astructural protein or enzyme; (ii) polynucleotides that are transcribedinto anti-sense molecules; (iii) polynucleotides that are transcribedinto decoys that bind transcription or translation factors; (iv)polynucleotides that encode cellular modulators such as cytokines; (v)polynucleotides that can make recipient cells susceptible to specificdrugs, such as the herpes virus thymidine kinase gene; and (vi)polynucleotides for cancer therapy, such as the wild-type p53 tumorsuppressor cDNA for replacement of the missing or damaged p53 geneassociated with over 50% of human cancers, including those of the lung,breast, prostate and colon.

Since the therapeutic specificity of the resulting recombinant AAVvector is determined by the plasmid introduced, the same packaging cellline can be used for any of these applications. The plasmid comprisingthe specific target polynucleotide is introduced into the packaging cellfor production of the AAV vector by one of several possible methods;including, for example, electroporation.

Helper virus can be introduced before, during or after introduction ofthe rAAV vector. For instance, the plasmid can be co-infected into theculture along with the helper virus. The cells are then cultured for asuitable period, typically 2-5 days, in conditions suitable forreplication and packaging as known in the art (see references above andexamples below). Lysates are prepared, and the recombinant AAV vectorparticles are purified by techniques known in the art.

In a preferred embodiment, also illustrated in the Examples below, therecombinant AAV vector is itself stably integrated into a packaging cellline. Such stable, vector-containing packaging lines can also optionallycontain stable chromosomal or episomal packaging cassettes. Cell linessuch as those described above can be grown and stored until ready foruse. To induce production of rAAV particles, the user simply infects thecells with helper virus and cultures the cells under conditions suitablefor replication and packaging of AAV (as described below).

Recombinant AAV vectors prepared using the methods and compositions ofthe present invention can be purified according to techniques known inthe art, see, e.g., the various AAV references cited above.Alternatively, improved purification techniques can be employed, such asthose described by Atkinson et al. in a commonly-owned U.S. applicationentitled Methods for Generating High Titer Helper-Free Preparations ofRecombinant AAV Vectors, filed 05 Sep. 1997 (as U.S. Ser. No.08/925,815).

The rAAV vectors can be used to deliver polynucleotides to target cellseither in vitro or in vivo, as described in the references cited hereinand in the art. For delivery in vivo, the rAAV vectors will typically becontained in a physiological suitable buffered solution that canoptionally comprise one or more components that promote sterility,stability and/or activity. Any means convenient for introducing thevector preparation to a desired location within the body can beemployed, including, for example, by intravenous or localized injection,by infusion from a catheter or by aerosol delivery.

EXAMPLES

A. Generation of an Integrated AAV Packaging Cassette for RAAVProduction

A-1. Construction of an AAV Packaging Cassette Employing P1 as anExemplary Activating Element

We have found that a P1 sequence, as found within a region believed tobe an AAV integration locus on human chromosome 19, can be used as anactivating element within the context of the present invention. Theexemplary P1 sequence we used comprises nucleotides 354468 of the AAV S1locus (Kelman et al (1994) Curr. Opin. Genet. Dev. 4:185-195 alsoWeitzman et al (1994) Proc. Natl. Acad. Sci. 91:5808-5817). Shown belowis the nucleotide sequence of P1 (SEQ ID NOs. 1 and 2), including apresumed terminal resolution site (TRS) at nucleotides 372-377, and apresumed Rep binding motif (RB Motif, also known as a Rep-binding siteor RBS), at nucleotides 386-401. Also indicated (by thedownward-pointing arrow) is the presumed Rep cleavage site locatedbetween the thymidines of the TRS.                        TRS                        → 5′CGGGCGGGTGGTGGCGGCGGTTGGGGCTCGGCGCTCGCTCGCTCGCTGGGCGGGCGGGCGGT 3′   |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 3′GCCCGCCCACCACCGCCGCCAACCCCGAGCCGCGAGCGAGCGAGCGACCCGCCCGCCCGCCA 5′                                      RB MotifA 2. Construction of p5Repcap

As an exemplary AAV packaging cassette, we linked a P1 element (asdescribed above) to AAV rep and cap genes that remained operably linkedto their native AAV promoters. As a first step in that process, an AAVpackaging cassette, p5repcap, comprising the AAV rep and cap encodingsequences transcriptionally linked to the native p5, p19 and p40promoters and followed by the AAV2 polyadenylation signal, wasconstructed as follows. Briefly, a fragment from pAV2 comprisingnucleotides 193 to 379 (Srivastiva et al. (1983) J. Virol. 45:555-564)was obtained by PCR amplification. The design of the PCR primersresulted in addition of a BglII site at the 5′ end of the amplifiedfragment and encompassed the PpuMI site (at AAV-2 nucleotide 350) closeto the 3′ end. The PCR-amplified DNA was digested with BglII and PpuMIto generate a restriction fragment comprising AAV-2 nucleotides 193-350.A restriction fragment comprising nucleotides 351-4498 of pAV2 wasisolated from pAV2 by digestion with PpuMI and SnaBI. These twofragments (representing nucleotides 193-4498 of pAV2) were ligated intoa tgLS(+)HyTK retroviral vector (S. D. Lupton et al., Molecular andCellular Biology, 11: 3374-33-78, 1991) in a four-way ligation that alsoincluded a StuI-BstEII fragment of tgLS(+)HyTK and a BstEI-StuI fragmentof tgLS(+)HyTK to which a BglII linker had been attached at the StuIend. This ligation generated tgLS(+)HyTK-repcap. Subsequently, a BglII-ClaI fragment from tgLS(+)HyTK-repcap, including AAV rep and cap genestranscriptionally linked to the native p5, p19 and p40 promoters andfollowed by the AAV2 polyadenylation signal, was isolated and clonedinto the BamHI and ClaI sites of pSP72 (Promega).

A 3. Construction of p5repcapDHFR

An AAV packaging expression plasmid, p5repcapDHFR, was constructed forthe purpose of producing an integrated packaging line including theconstruct p5repcap (Example A 2) and a modified dihydrofolate reductasegene (DHFR) as a selectable marker. Specifically, p5repcap (Example A 2)was linearized at a PvuII site located just upstream of the rep gene,and blunt-end ligated to a 1.8 kb fragment of pFR400 (Simonsen et al.(1983) Proc. Natl. Acad. Sci. USA 80:2495-2499). This pFR400 fragmentwas comprised of a modified DHFR gene, with a reduced affinity formethotrexate (Mtx), transcriptionally linked to the SV40 early promoterand followed by the polyadenylation site from the Hepatitis B virus(HBV) surface antigen gene. The pFR400 fragment was prepared by firstdigesting with SalI, followed by a four base pair fill-in (to generate ablunt end) and subsequent PvuII digestion and gel purification. Theresulting construct, p5repcapDHFR (FIG. 1), contains a DHFR gene whosetranscription is regulated by an upstream SV40 early promoter and adownstream Hepatitis B Virus polyadenylation site. Immediatelydownstream of this DHFR transcriptional cassette lie the AAV rep and capgenes, followed by an AAV polyadenylation site.

A 4. Addition of P1 to a Repcap-Containing Plasmid: Construction ofP1RCD

An exemplary AAV packaging cassette was then generated by incorporatinga P1 element (Example A 1) into expression plasmid p5repcapDHFR (ExampleA 3). In the construction of the plasmid, “P1RCD”, containing thispackaging cassette, the P1 element was inserted downstream of the AAVpolyadenylation signal in p5repcapDHFR in an orientation such thatreplication initiating from the P1 element proceeds first into the capgene and then into the rep gene (i.e., replication initiates at the3′-OH of the TRS on the anti-sense strand and proceeds in a 5′-to-3′direction towards the cap gene). To facilitate insertion of the P1element into p5repcapDHFR, a pair of oligonucleotides were synthesizedwhich include the P1 sequence flanked by ends compatible with a BglIIrestriction site (see sequences below, SEQ ID NOs. 3 and 4). The pairwere annealed, then ligated to p5repcapDHFR previously linearized at aBglII site located just downstream of the AAV polyadenylation site(Example A 3, nucleotide 6217). A clone named P1RCD was selected,containing a P1 insert in an orientation such that replication initiatedat P1 proceeds in the direction of the cap and rep genes (FIG. 2).        P1 Oligonucleotide pair:                 Top line: SEQ ID NO 3                                              Bottom line: SEQ ID NO 4                              RB Motif 5′GATCACTAGTACCGCCCGCCCGCCCAGCGAGCGAGCGAGCGCCGAGCCCCAACCGCCGCCACCACCCGCCCGA3′       |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||3′     TGATCATGGCGGGCGGGCGGGTCGCTCGCTCGCTCGCGGCTCGGGGTTGGCGGCGGTGGTGGGCGGGCTCTAGA5′                                                     TRS

Additional exemplary constructs were produced in which the location andmultiplicity of the P1 element was varied. P1(5′)RCD contained a singleP1 element upstream of the rep and cap genes at a distance of 1.5kilobases from the rep translation initiation site. The construct 2P1RCDcontained two P1 elements: the first located immediately downstream ofcap as in P1RCD (see above) and the second inserted 1.5 kb upstream ofrep as in P1(5′)RCD described above.

Insertion of a P1 element into p5repcapDHFR to generate P1(5′)RCD, andinto P1RCD to generate 2P1RCD, was performed in a manner analogous tothat described above for insertion of a P1 element into p5repcapDHFR togenerate P1RCD, except that the oligonucleotide pair listed below (SEQID NO 5) was used. The new oligo pair was annealed and ligated intop5repcapDHFR and P1RCD previously linearized at the Pvu II site located1.5 kilobases upstream of the rep translation initiation codon. Cloneswere selected such that the orientation of the P1 insert resulted inDNA-replication proceeding first into the rep gene and then into cap.

P1 aligo pair for construction of P1(5′)RCD and 2P1RCD SEQ ID NO 5:                       TRS 5′CCCGGGCGGGTGGTGGCGGCGGTTGGGGCTCGGCGCTCGCTCGCTCGCTGGGCGGGCGGGCGGTCAG 3′   |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||3′ GGGCCCGCCCACCACCGCCGCCAACCCCGAGCCGCGAGCGAGCGAGCGACCCGCCCGCCCGCCAGTC5′                                      RB Motif

Additional exemplary packaging plasmids were constructed that containedP1 elements in constructs lacking a selectable marker. ConstructP1(5′)RC contained a single P1 element immediately upstream of the repand cap genes; P1RC contained a single P1 element immediately downstreamfrom the rep and cap genes; and 2P1RC contained two P1 elements flankingrep-cap. The constructs were produced as described above in this exampleexcept that AAV packaging construct p5repcap (Example A2) was used inplace of AAV packaging construct p5repcapDHFR. The P1 sequence wasinserted as described above in this example using both of the oligopairs described above, as appropriate. Virus was produced byco-transfection of either p5repcap, P1RC, P1(5′)RC, or 2P1RC along withrAAV vector ACAPSN according to the method of Example A6, infra. Virustiter was measured for each using the method of Example A7, infra.

A 5. Construction of rAAV Vector ACAPSN

The plasmid ACAPSN was constructed according to Lynch et al. (1997)Circ. Res. 80: 497-505 and PCT Publication WO 97/32990, as follows. TheITR sequences and plasmid backbone were derived from AAV-CFTR. Afione etal. (1996) J. Virol. 70:3235-3241. Briefly, the AAV-CFTR vector wasdigested with XhoI and SnaBI and the ITRs and plasmid backbone were gelisolated. An XhoI to SnaBI fragment containing a portion of the CMVpromoter (nucleotides −671 to −464) [See, e.g., Boshart, et al., Cell,41: 521-530 (1985)] was gel isolated and ligated to the ITR plasmidbackbone fragment derived from AAV-CFTR to generate “pAAV-CMV (SnaBI).”Next, an SpeI to SnaBI fragment containing the synthetic polyadenylationsignal was inserted into SpeI/SnaBI digested pAAV-CMV (SnaB1) togenerate “pAAV-CMV (SpeI)-spA.” The pAAV-CMV (SpeI)-spA vector containsnucleotides −671 to −584 of the CMV promoter. Next, the human placentalalkaline phosphatase cDNA sequence linked to the Simian virus 40promoter driving the E. coli neomycin gene was isolated from LAPSN [See,e.g., Clowes et al. (1994) J. Clin. Invest. 93: 644-651] as an SpeI toNheI fragment and inserted into pAAV-CMV (SpeI)-spA (which had beenlinearized with SpeI) to create “pAAV-APSN.” An SpeI to NheI fragmentcontaining CMV promoter nucleotides −585 to +71 was inserted intoSpeI-linearized pAAV-APSN to generate vector “ACAPSN.”

A 6. Virus Production

Packaging of rAAV particles was performed as previously described. See,e.g., Flotte et al., J. Biol. Chem. 268 (5): 3781-3790 (1993); Flotte etal., Proc. Natl. Acad. Sci. USA, 93: 10163-10167 (1993); and Flotte etal. (1995) Gene Ther. 2:29-37. According to these protocols, equalamounts of packaging plasmids (either p5repcapDHFR or P1RCD) and therAAV vector ACAPSN were co-transfected into HeLa cells which had beeninfected with helper Ad 5 at a MOI of 5. After incubation for 65 hoursat 37° C. in a humidified atmosphere of 10% CO₂, cells were harvestedand lysed by freeze/thawing and sonication. Cell debris was removed bycentrifugation at 3000×g for 5 minutes. The resulting cleared lysateswere heat-treated for 1 hour at 56° C. to inactivate residualadenovirus.

A 7. Measurement of Virus Titer by G418 Resistance

Methods The titer of virus produced by the method in Example A 6 fromco-transfection of ACAPSN and either the p5repcapDHFR or P1RCD AAVpackaging plasmid was determined by the measurement of geneticin (G418)resistance. The protocol includes seeding 5×10⁴ HeLa cells per well in a6 well dish (Costar) in Dulbecco's Modified Eagles medium, 10% fetalbovine serum, with penicillin and streptomycin (DMEM complete). After 24hours, cells were exposed to serial dilutions (in DMEM) ofvirus-containing cleared lysates (Example A 6) for 24 hours at 37° C. ina total volume of 1 ml (the maximal amount of cleared lysate that isassayable being 0.1 ml). Virus-containing medium was then removed andfresh DMEM, containing 1 mg/ml G418, was added to the cells. Cells werecultured for 10 days under selective conditions, medium was thenremoved, and the cells were washed once in methanol and stained withmethylene blue. Colonies on each well were then counted and resultsexpressed as G418-resistant colony forming units per milliliter(G418^(r) cfu/ml).

Results The packaging plasmids P1(5′)RCD, P1RCD and 2P1RCD (see ExampleA 4) were assayed for their ability to produce virus in aco-transfection with rAAV vector ACAPSN. Co-transfection, helper virusinfection and preparation of cleared lysates were performed as describedin Example A6.

The construct containing a single P1 element downstream of cap (P1RCD)produced four-fold more virus than the non-P1 containing construct,p5repcapDHFR (1900+/−1400 cfu/mL vs. 490+/−58 cfu/mL, respectively).When the P1 element was located at a distance of 1.5 kb upstream of therep-cap gene cassette (P1(5′)RCD), a 20-fold increase in virusproduction was observed relative to the non-P1 construct, p5repcapDHFR(9900+/−1000 cfu/mL vs 480+/−58 cfu/mL, respectively). Incorporatingboth P1 elements, such that one was located 1.5 kb upstream of rep andthe other was immediately downstream of cap, resulted in a furtherincrease in virus production (17,500+/−2000 cfu/mL), i.e. 36-foldcompared to p5repcapDHFR.

The AAV packaging constructs lacking a DHFR marker containing a singleP1 element either immediately upstream or downstream of the rep and capgenes (P1(5′)RC or P1RC, respectively, see Example A 4) resulted in a 3fold increase in rAAV vector titer compared to the non-P1 containingconstruct, p5repcap (5500+/−1514 Cfu/ml or 5700+/31 1172 vs. 1700+/−560Cfu/ml). Incorporating both P1 elements flanking the rep and cap genes(2P1RC, see Example A 4) further increased virus production 10-foldcompared to the single P1-containing constructs (53000+/−8082 Cfu/ml),equivalent to a 30-fold increase in viral titer compared to the non-P1containing construct, p5repcap. These results show that P1 functions toamplify vector production, independent of location or distance from therepcap gene cassette, when tested in transient co-transfection.

A 8. Production of Packaging Cell Lines

Polyclonal cell lines with an integrated AAV packaging cassette eithercontaining (P1RCD) or lacking (p5repcapDHFR) the P1 element wereproduced by electroporation of HeLa cells. Specifically, 4×10⁶HeLa cellswere electroporated with 12 μg DNA (p5repcapDHFR or P1RCD) that had beenlinearized with PvuII restriction endonuclease, which cleaves justupstream of the SV40 promoter-DHFR gene cassette. The cells wereelectroporated in serum free DMEM using a BioRad Gene Pulser at 0.25Volts and 960 μF. After electroporation, cells were resuspended in DMEMcomplete (see Example A 7) and allowed to recover at 37° C. in ahumidified atmosphere of 10% CO₂. After 24 hours, cells were subjectedto selection in complete medium containing 500 nM methotrexate. Clonalcell lines were derived from the P1RCD polyclonal population by limitingdilution. Producer lines are generated by introduction of an rAAV vectorconstruct into a clonal P1RCD-containing packaging line.

The constructs p5repcap, P1RC, P1(5′)RC, and 2P1RC (see Example A 4)were modified for the purpose of producing stable cell lines byfollowing the procedure described in Example A 3, using a puromycinresistance gene in place of the modified DHFR gene. The four resultingAAV packaging constructs were named p5RC-Pur, P1RC-Pur, P1(5′)RC-Pur,and 2P1RC-Pur. Polyclonal cell lines were produced from these fourconstructs as described above in this example, except the methotrexateselection was replaced with drug selection by puromycin at aconcentration of 1 μg/mL.

A 9. Isolation of Total Genomic DNA from Packaging Cells

rAAV genomes were packaged according to Example A 6 in polyclonal celllines containing either p5repcapDHFR or P1RCD (Example A 8) bytransfection with ACAPSN in the presence or absence of adenovirus. At 65hours after transfection with ACAPSN, cells were harvested andcentrifuged at 3000×g for 5 minutes. Total genomic DNA was isolatedaccording to the method previously reported (Sambrook et al., supra).Specifically, cells were washed once with TBS (150 mM Trizma base, 300mM NaCl, pH 7.4) and resuspended in TNE Buffer (10 mM Tris-Cl pH 8, 100mM NaCl and 25 mM EDTA pH 8). Proteinase K was added to a finalconcentration of 100 μg/ml and SDS was added to a final percentage of0.5% (w/v). After mixing, cells were incubated at 50° C. for 3 hours.Samples were then extracted once with phenol (pH 8), once withphenol:chloroform:isoamyl alcohol (24:24:1), and once with chloroform.DNA, present in the aqueous phase, was then precipitated with 100%ethanol and centrifuged at 12,000×g for 30 minutes. The pellets,containing genomic DNA, were washed once with 70% ethanol, air dried,and resuspended in TE buffer (10 mM Tris, 1 mM EDTA pH 8).

A 10. Southern Blotting Analysis

Total genomic DNA isolated by the method of Example A 9 was examined forthe amplification of rep and cap genes in the presence and absence ofadenovirus. Specifically, 10 μg of DNA was digested with restrictionendonuclease BglI thereby releasing a 3.8 kb fragment comprising rep andcap genes (AAV-2 nucleotides 543-4,380) from p5repcapDHFR or P1RCD.Digested DNA samples were then fractionated by agarose gelelectrophoresis and transferred to UV-Duralon membrane (Stratagene) bycapillary action, overnight, in 10×SSC (1.5 M NaCl, 0.15M SodiumCitrate). Nucleic acid was cross-linked to the membrane by exposure toultraviolet light, and the membranes were rinsed in 2×SSC and probedwith a ³²P labeled 1.9 kb XhoI-BglII fragment from pA V2, random-primelabeled using prime-it, Stratagene. After washing, the membranes werevisualized by phosphorimaging and the amount of the 3.8 kb band wasquantified.

A 11. Analysis of Packaging Cassette Amplification in PolyclonalPackaging Cell Lines

Total genomic DNA prepared and digested according to Example A 9 forpolyclonal samples P1RCD and p5repcapDHFR (Example A 8) was analyzed bythe Southern blotting method of Example A 10. Degree of amplificationwas measured by relative photon intensity of the 3.8 kb band determinedfrom phosphorimaging according to Example A 10. DNA fromP1RCD-containing cells gave a value of 406,725 intensity units for the3.8 kb band, while DNA from cells containing p5repcapDHFR gave a valueof 30,211. Thus the presence of P1, in the P1RCD polyclonal line, isresponsible for a 13.5-fold amplification of rep and cap genes, in thepresence of adenovirus.

A 12. Virus Production by Packaging Cell Lines

Polyclonal cell lines, containing either P1RCD or p5repcapDHFR, weretransiently transfected with ACAPSN in the presence of adenovirus, rAAVgenomes were packaged, and cleared lysates were produced according tothe method of Example A 6. Cleared lysates were assayed for viral titer(Example A 7), which was determined from triplicate transfections. Whenmaximal amounts of cleared lysate were assayed (i.e., the amount atwhich non-specific cell killing begins to occur), a polyclonal cell linecontaining p5repcapDHFR yielded 0 G418^(r) cfu/ml, while a polyclonalcell line containing P1RCD yielded 957 G418^(r) cfu/ml. Virus productionby clonal lines ranged from 0.8×10²−1.5×10⁴ G418^(r) cfu/ml.

Stable cell lines containing integrated P1-containing packaging plasmidsexpressing puromycin resistance were tested for virus production. Astable cell line containing a single P1 element downstream from the capgene (P1RC-Pur) increased virus titer 4 fold over the non-P1 containingcell line, p5RC-Pur (216+/−67 Cfu/ml vs. 51.1+/−30 Cfu/ml). When the P1element was located upstream of the rep gene (P1(5′)RC-Pur), a similarincrease in virus titer occurred (333 Cfu/ml +/−150), 6 fold over thenon-P1 containing cell line, p5RC-Pur. The stable cell line containing 2P1 elements flanking rep-cap (2P1RC-Pur), resulted in a further increasein viral titer (658+/−122 Cfu/ml) to 13-fold that of the non-P1containing cell line, p5RC-Pur. These results show that P1 functions toamplify vector production in a stable cell line, regardless of itslocation.

B. Use of an Episomally-Maintained AAV Packaging Cassette for RAAVProduction

B 1. Construction of an EBNA-1 Based AAV Packaging Vector Containing P1Elements

The EBNA-1 episomal packaging cassette containing AAV rep and cap genesalong with two P1 elements was constructed in the following manner. Two69 bp oligonucleotides containing the published P1 sequence (Urcelay etal. (1995) J. Virol. 69:203846) were synthesized. In addition to the P1sequence, the oligonucleotides contain a unique Smal restriction site.After annealing, the SphI-compatible oligonucleotides were inserted intothe SphI site of the p5repcap vector (Example A 2). A clone containingtwo opposing concatameric P1 elements (TRS-RBS-RBS-TRS) was obtained.

A 4424 bp PvuII/BglII fragment containing the two P1 elements andp5repcap sequences was isolated. These sequences were inserted into theNruI/BamHI-digested Rep8 EBNA-1 plasmid (Invitrogen). The resultingplasmid was designated P1/p5repcap/Rep8. In this construct, the tandemP1 sequences are located 84 nucleotides upstream of the p5 promoter andthe associated rep and cap genes (FIG. 3).

In addition, a p5repcap/Rep8 plasmid that did not contain a P1 elementwas constructed by isolating a 4355 bp PvuII/BglII fragment from thep5repcap vector. This fragment was inserted into NruI/BamHI digestedRep8. The resulting plasmid was designated p5repcap(-P1)/Rep8.

B 2. Generation of Cell Lines Containing a Stably-Integrated rAAV Vectorand an Episomal P1 AAV Packaging Cassette

The rAAVCFTR or ACAPSN vector was transfected into HeLa cells viaelectroporation. Individual clones were isolated and screened for anintact, stably integrated rAAV vector. The P1/p5repcap/Rep8 packagingcassette was then transfected into HeLa/AAV-CFTR cells viaCaHPO₄-mediated transfection and stable transfectants were selectedusing 2.5 mM L-histidinol. A HeLa/AAV-CFTR cell line containing ap5repcap(-P1)/Rep8 packaging cassette was generated in similar fashion.

B 3. Amplification of P1-Containing Episomal Packaging Cassette

To determine if the P1/p5repcap/Rep8 packaging cassette is amplifiablein stable HeLa/AAV-CFTR cell line, the following experiment was carriedout. The stable HeLa/AAV-CFTR cell line from Example B2 were seeded induplicate at 2.5×10⁵ cells/plate. After 24 hrs one plate for each cellline was infected with Ad5 at a multiplicity of 10. After 48 hrs.infected and uninfected cells were harvested. The genomic DNA wasisolated, digested with BglII and XbaI restriction enzymes, and theresultant fragments were separated by electrophoresis and transferred toa membrane. The blot was then probed with a ³²P-labeled 2.0 kb repfragment and the degree of amplification was determined by Southernblotting as described in Example A 10. The results are shown in FIG. 4and indicate that in the presence of adenovirus the P1-episomalpackaging cassette exhibits a high degree of amplification (10 to 100fold increase), whereas in the absence of adeno virus no amplificationis observed. The p5repcap(-P1) episomal packaging cassette exhibitedvery little detectable amplification in the presence and no detectableamplification in the absence of adenovirus. B4. Production of rAAV-CFTRVector in HeLa/AAV-CFTR Cell Line Containing a P1/p5Repcap/Rep8Packaging Cassette

To demonstrate rAAV virus production in a HeLa/AAV-CFTR cell linecontaining a P1/p5repcap/Rep8 episomal packaging cassette, the followingexperiment was carried out. See PCT Publication WO 96/17947 for details.Briefly, HeLa/AAV-CFTR cells containing either a P1/p5repcap/Rep8 or ap5repcap(-P1)/Rep8 episomal packaging cassette were seeded at 2.5×10⁶cells/plate and infected with Ad5 at a MOI of 10. After 48 hrs., thecells were harvested, resuspended in TMEG buffer, and sonicated in15-second bursts for 2 min. to release rAAV. One percent of the crudelysate was heat-treated at 56° C. for 45 min and then added to 2.5×10⁵cl.37 cells, with or without Ad5. The cells were harvested after 48 hrsand genomic DNA was isolated. The DNA was digested with EcoRI, resolvedby electrophoresis, transferred to a membrane and probed with a³²p-labeled 1.4 kb CFTR fragment. Results are shown in FIG. 5 andindicate that in the presence of Ad5 the cell line containing theP1/p5repcap/Rep8 packaging cassette was producing at least 10 times morevirus than the cell line containing the p5repcap(-P1)/Rep8 packagingcassette.

B5. P1-EBNA Vector Variations

To reduce the potential for generating wild type AAV orreplication-competent chimeric AAV, a second generation P1/p5repcap/Rep8packaging cassette was constructed that contains a nonessential 1300 bpDNA stuffer fragment between the P1 elements and the p5repcap sequences.To construct this packaging cassette; a 4355 bp PvuII/EcoRV fragmentcontaining p5repcap sequences was isolated from the p5repcap vector(Example A 2). This fragment was inserted at the EcoRV site of pAdBn(Quantum Biotechnologies). The resulting plasmid was digested with BglIIand NotI and a 4485 bp p5repcap fragment was isolated. This BglII/NotIfragment was inserted into BamHI/NotI-digested pRep8 (Invitrogen).

To insert the two concatameric P1 elements into this plasmid theP1/p5repcap/Rep8 packaging cassette (see Example B 1) was digested withPstI to remove the p5repcap sequences. The plasmid backbone, containingthe two P1 elements, was religated. The resulting plasmid was digestedwith PvuII and NotI, and a 138 bp fragment, containing concatameric P1sites, was isolated. This P1 dimer was then inserted intoP1/p5repcap/Rep8 that had been digested with NruI and NotI; Finally, toinsert the nonessential stuffer fragment, a 1300 bp HaeIII fragment fromΦX174 was ligated into the SnaI site of P1/p5repcap/Rep8. The resultingplasmid is used as an AAV packaging cassette to stimulate replicationand packaging of rAAV vectors.

While the invention has been described, for purposes of clarity andillustration, with reference to the description and examples above, itis clear that many variations and modifications can be made by one ofskill in the art, without departing from the scope of the appendedclaims.

1. A recombinant polynucleotide sequence encoding an adeno-associatedvirus (AAV) packaging cassette comprising at least one AAV packaginggene amplifiably linked to an activating element.
 2. The packagingcassette of claim 1 wherein the AAV packaging cassette comprises an AAVrep gene and an AAV cap gene.
 3. The packaging cassette of claim 1wherein the activating element is a P1 element.
 4. The packagingcassette of claim 1 wherein the activating element is an induciblereplication origin.
 5. The packaging cassette of claim 1 wherein theactivating element is a replication origin that is activated by helperfunction.
 6. The packaging cassette of claim 5 wherein the helperfunction is provided by adenovirus.
 7. The packaging cassette of claim 1wherein the activating element comprises a viral replication origin. 8.The packaging cassette of claim 7 wherein the viral replication originis dependent on helper function.
 9. The packaging cassette of claim 8wherein the helper function is provided by adenovirus.
 10. The packagingcassette of claim 1 wherein the activating element comprises a mammalianreplication origin.
 11. The packaging cassette of claim 2 wherein theactivating element comprises a sequence having activity in a replicationassay.
 12. The packaging cassette of claim 11 wherein the replicationactivity is Rep-dependent.
 13. The packaging cassette of claim 1,wherein said activating element comprises a Rep Binding Motif and aTerminal Resolution site.
 14. A method for producing high-titer stocksof an rAAV vector containing a heterologous gene of interest, comprisingco-expressing the rAAV vector containing a gene of interest in amammalian cell along with an AAV packaging cassette, said AAV packagingcassette comprising at least one AAV rep and/or cap gene amplifiablylinked to an activating element.
 15. The method of claim 14 wherein theactivating element is a P1 element.
 16. The method of claim 14 whereinthe activating element is a replication origin that is activated byhelper function.
 17. The method of claim 16 wherein the helper: functionis provided by adenovirus.
 18. The method of claim 14 wherein theactivating element is an inducible replication origin.
 19. A method forgenerating a cell line capable of producing high-titer stocks of an rAAVvector containing a foreign gene of interest, by transfecting mammaliancells with an rAAV vector containing a gene of interest and with an AAVpackaging cassette, said AAV packaging cassette comprising at least oneAAV rep and/or cap gene amplifiably linked to an activating element. 20.The method of claim 19 wherein the activating element is a P1 element.21. The method of claim 19 wherein the activating element is areplication origin that is activated by helper function.
 22. The methodof claim 21 wherein the helper function is provided by adenovirus. 23.The method of claim 19 wherein the activating element is an induciblereplication origin.
 24. An AAV packaging cell line produced by themethod of claim
 23. 25. An AAV virus containing an rAAV vector producedby the method of claim 14.